Project Summary Congenital hyperinsulinism (HI) is the most frequent cause of persistent hypoglycemia in infants and children. Children with HI are at high risk of seizures and permanent brain damage and treatment of their hypoglycemia is extremely difficult. Recent work has shown that HI is associated with genetic defects in the pathways regulating beta-cell insulin secretion. Although 6 such loci have been found, many children with HI have no identifiable mutation of these genes. This includes one-third of diffuse HI cases that require pancreatectomy and half of cases that are responsive to medical treatment with diazoxide. Our hypothesis is that hyperinsulinism in these groups of children involves both novel molecular defects of known loci, as well as, previously unrecognized new genetic loci. Our long-term goal is to identify genotype-phenotype correlations in these disorders to guide diagnosis and treatment and to uncover new forms of congenital hyperinsulinism. Aim 1 will identify the novel genetic locus in the historically-important dominant HI family reported by McQuarrie in 1954. Preliminary data have mapped this form of HI to an 8.4 mb region on chromosome 10q that includes the high affinity hexokinase enzyme, HK1. The phenotype of islet dysregulation in affected family members will be defined by clinical studies of insulin secretion. Linkage analysis and newly-available gene capture and high- throughput sequencing methods will be used to identify the disease-causing mutation responsible for this novel form of HI. Aim 2 will search for defects in novel candidate genes in our large group of children with diazoxide- responsive hyperinsulinism that have no identifiable mutation. Mutation analysis of peripheral blood genomic DNA will be used to examine potential candidate genes, including the novel gene on 10q that will be identified in Aim 1, HADH/SCHAD, SLC16A1/MCT-1, TCF1/HNF1, TCF4/HNF4, SIR2L4/SIRT4, and others. Aim 3 will define the mechanisms of molecular defects in children who fail to respond to diazoxide and require pancreatectomy. We will search for novel molecular defects of the two adjacent genes on 11p that are responsible for most cases of this form of HI: ABCC8/SUR1 and KCNJ11/Kir6.2. This will include mutation analysis of the entire 120 kb region of these genes using high-throughput sequencing; analyzing pancreatic tissue for genetic and histological evidence of post-zygotic, mosaic mutations; testing for mutations in promoter regions, 3'UTR regions, and microRNA sites; and screening for epigenetic methylation defects.